Method for preparing organic, in particular lithocholic acid, nanotubes and uses thereof

ABSTRACT

The invention relates to a method for preparing organic nanotubules in an aqueous medium, comprising the following steps:  
     a) preparing a basic aqueous solution with a pH from 10 to 14, preferably from 12 to 13.5,  
     b) adding to the solution an organic compound of formula:  
                 
 
     wherein R 1  is a C 17 -C 20  polycyclic radical with fused rings optionally including alkyl substituents, R 2  is a C 3 -C 20  linear or branched alkylene group, and R 3  represents a hydrogen atom, a C 1 -C 20  alkyl or C 6 -C 30  aromatic group, and  
     c) submitting the solution to stirring for sufficient time in order to form stable tubules of the organic compound in the solution.  
     The organic compound may be lithocholic acid.

TECHNICAL FIELD

[0001] The object of the present invention is the manufacturing oforganic nanotubules.

[0002] Such nanotubules may find various applications, notably in thesector of biosensors and carriers of active ingredients, and in thenanoelectronics sector.

STATE OF THE PRIOR ART

[0003] Spontaneous self-assembly of small molecules into nanostructuresis a mild and simple alternative method for manufacturing materials, theactivity center of which is also located at a nanoscopic scale. Twolarge application sectors may be concerned by these nanotubularstructures, as described by Bong, D. T., Clark, T. D. Granja, J. R. andGhadiri, M. R. (2001), Angew. Chem. Int. Ed., 40, 988-1011, [1].

[0004] The first sector is that of chemistry where these nanotubularstructures may be used in biosensors or for transporting bioactiveproducts.

[0005] The second sector is that of electronics and electro-optics, asdescribed by Trau M., Yao N., Kim E., Xia Y., Whitesides, G. M. andAksay I. A. (1997), Nature, 390, 674-676 [2].

[0006] Schematically, the functioning of applications in the firstsector may be summarized by showing that the tubules may be used as suchif the medium where they exist is compatible with the one to be probedor treated. Diffusion limited by the size of the hollow core of thetubule may be the operational principle of one of these types ofapplications. If this core may be suitably functionalized so as toexhibit electronic and/or ionic conduction properties as described byVan Nostrum, C. F. (1996), Adv. Mater., 8, 1027-1030 [3], otherapplications are possible.

[0007] The second field of application proceeds with the stiffening ofthe tubules in order to make a solid replicate which in turn may be usedfor the aforementioned applications, but also for manufacturingelectro-active composite materials. This stiffening step may be achievedby metallization of the structures or mineralization with silica. At thesame time, electromagnetic properties of interest for the applicationsmay be introduced by metallization. Mineralization is a mild chemistrymethod providing a transition from the organic domain to the mineraldomain, fields of application for carbon nanotubes as described byDekker, C. (1999), Physics Today, 52, 22-28 [4] may be rediscovered withstiffened tubules as described by Schnur, J. M. (1993), Science, 262,1669-1676 [5], and Schnur, J. M. and Shashidhar, R. (1994), Adv. Mater.,6, 971-974 [6].

[0008] As for applications outside the electronics and opto-electronicsfield, catalysis, biomolecular separation and the preparation of porousmembranes with calibrated holes for filtration applications where areplica is made by polymerization around fibrillar structures asdescribed by Gu, W., Lu, L. Chapman, G. B. and Weiss, R. G. (1997) J.Chem. Soc., Chem. Commun., 543-544 [7], may be also mentioned. Othersilica-based structures have been described by Jung, H. J. Amaike, M.and Shinkai S. (2000), J. Chem. Soc., Chem. Commun., 2343-2344 [8].

[0009] Very often, the effectiveness of a system formed from nanoscopicindividual active components will be enhanced if these components areorientated and aligned.

[0010] Presently, chemical systems leading to the formation of tubulesbelong to the following chemical classes described in reference [1]:

[0011] linear or cyclic peptides,

[0012] macrocycles,

[0013] cyclodextrins,

[0014] lipids, and

[0015] block copolymers.

[0016] With these molecules, the internal diameter of the tubules is notalways in the nanometer range, between a few tens of angstroms,({fraction (1/10)}^(th) of a nanometer, i.e. 10⁻⁹ m) and few hundreds ofangstroms. Moreover, these molecules result from sometimes complexchemical syntheses.

[0017] The manufacturing methods often involve a heating step andsometimes several constituents.

[0018] Moreover, the orientation of the tubules assumes that they weaklyinteract with each other and then the use of electric fields isrequired. In other cases, orientation is achieved by usingmicrocapillaries as described in reference [7].

[0019] Thus, in reference [2], tubules may be obtained fromself-assembly of fibers full of cetyltrimethylammonium chloride. Theirorientation is achieved by applying an electric field inmicrocapillaries where the chemical reaction for forming the tubules andsimultaneous mineralization are conducted. Here, the alignment procedureis therefore complex. The internal diameter of the resulting tubule isset by the diameter of the initial full fibers; the external diametercannot be monitored accurately. The starting product is a syntheticsurfactant.

[0020] In reference [8], silica tubules are obtained via a sol-gelpolymerization technique on organic structures.

[0021] In references [5] and [6], tubules may be formed from thepolymerizable phospholipid,1,2-bis(tricosa)-10,12-10-diynoyl)sn-glycero-3-phosphocholine which isnot a simple chemical structure. The internal diameter is of the orderof 0.2 to 0.7 μm and is not located in the nanoscopic range.

[0022] The methods and molecules used hitherto for forming tubules thushave certain drawbacks.

[0023] Indeed, they do not meet the whole set of sought-after featuresfor obtaining such structures, which are:

[0024] 1) the ease in obtaining the starting product in order to avoidcomplex and costly chemical syntheses;

[0025] 2) the ease in obtaining tubules from the starting product;

[0026] 3) the homogeneity of the formed structures, i.e., the dimensionsof the external diameter and of the internal hole which must beperfectly calibrated;

[0027] 4) the stability of the formed nanostructures; and

[0028] 5) the ease in obtaining tubules aligned in parallel with eachother.

DISCUSSION OF THE INVENTION

[0029] Specifically, the object of the present invention is a method forobtaining tubules for which the dimensions of the external diameter andof the internal diameter, i.e., of the internal hole, are perfectlycalibrated in the nanometer range, by using as a starting product, anaturally secreted bile product and which does not require any furtherorganic synthesis operation, and with which nanotubules may easily beobtained in an aqueous medium.

[0030] According to the invention, the method for preparing organicnanotubules in an aqueous medium comprises the following steps:

[0031] a) preparing a basic aqueous solution with a pH from 10 to 14,preferably from 12 to 13.5,

[0032] b) adding to the solution an organic compound of formula:

[0033] wherein R¹ is a C₁₇-C₂₀ polycyclic radical with fused rings,optionally including one or more C₁₇-C₂₀ alkyl substituents, R² is aC₃-C₂₀ linear or branched alkylene, and R³ represents a hydrogen atom, aC₁-C₂₀ alkyl or a C₆-C₃₀ aromatic group, and

[0034] c) submitting the solution to stirring for sufficient time inorder to form stable tubules of the organic compound in the solution.

[0035] The starting organic compound used in the invention thuscomprises a carboxyl and hydroxyl group separated by a polycyclicoptionally substituted radical with fused rings and an alkylene groupwith which tubules of calibrated dimensions may easily be formed.

[0036] The R² group of this compound is an alkylene group which mayinclude from 3 to 20 carbon atoms, preferably from 3 to 8 carbon atoms.

[0037] As an example of such a group, the group:

[0038] may be mentioned.

[0039] In these compounds, the R¹ group is a polycyclic group with fusedrings, which may be derived from natural products such as steroids.

[0040] In this compound, the dimensions of the tubules, i.e., theirinternal and external diameters may be adjusted in particular byselecting groups R¹ and R².

[0041] As examples of such polycyclic groups, the group of formula:

[0042] may be mentioned, which may include one or several C₁ to aboutC₂₀ alkyl substituents.

[0043] According to the invention, R³ may represent a hydrogen atom, aC₁-C₂₀ linear or branched alkyl, or a C₆-C₃₀ aromatic group.

[0044] By aromatic group, a group is meant having one or more benzene,naphthalene, anthracene rings, and which may optionally includeheteroatoms such as O, S and N.

[0045] As examples of such aromatic groups, groups derived fromanthracene, benzene and naphthalene may be mentioned, such as anthryl,benzyl and naphthyl groups, and groups derived from azobenzene.

[0046] Preferably, according to the invention, the starting organiccompound is lithocholic acid of formula:

[0047] According to the invention, this acid which is normally insolublein water at a neutral or acid pH, is solubilized as stable nanotubulesby using a basic medium with a suitably adjusted pH. By simply stirringthe medium, nanotubules with monodispersed dimensions are obtained andare a signature of the initial constituent. The tubules may beorientated by simple shearing of the solution.

[0048] Thus, the method of the invention does not require any heating,and uses a natural starting product which does not require any furtherorganic synthesis operation; it further does not require the use ofelectrical fields or micro-etchings for orientating the tubules; ittherefore meets the whole set of sought-after features for obtaining thetubules.

[0049] According to the invention, the aqueous medium is a basic medium,preferably with a pH from 12 to 13.5. An aqueous sodium hydroxidesolution may be used. When the starting compound is lithocholic acid, asodium hydroxide solution is preferably used with a pH of about 12.5.

[0050] Adjustment of the pH of this medium, as well as the nature of thebase used (ionic force, counter-ions), and the concentration of organiccompounds, are also a means for adjusting the amplitude of theinteractions between tubules.

[0051] Preferably, this organic compound concentration is from 0.01% to20% by weight.

[0052] According to the invention, the kind of polycyclic radical R¹ andthe aqueous medium (light water, heavy water) also play a role on thestructural features of the tubules.

[0053] The object of the invention is further the use of organicnanotubules obtained by this method for manufacturing inorganicnanotubes by mineralization, for manufacturing field emission cathodesby metallization of nanotubes or furthermore for manufacturingbiosensors.

[0054] SL

[0055] Other features and advantages of the invention will become moreapparent upon reading the following description, naturally given asillustrative and non-limiting, with reference to the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 illustrates typical X-ray scattering from a system oflithocholic acid tubules in a basic medium.

[0057]FIG. 2 is an electron micrograph of lithocholic acid tubules.

[0058]FIG. 3 illustrates the curves of stress σ (in Pa) versus shearrate {dot over (γ)} or dγ/dt (in s⁻¹) for two solutions of lithocholicacid tubules.

DETAILED DISCUSSION OF AN EMBODIMENT

[0059] An example of the preparation of lithocholic acid tubules isdescribed hereafter.

[0060] First of all, a basic aqueous solution is prepared with a pH ofabout 12.5, by adding 0.125 g of sodium hydroxide to 100 g of water.Next, 0.3 grams of lithocholic acid are added to the obtained solution.The whole solution is submitted to stirring by means of a magneticstirrer for about half an hour and a solution of tubules at aconcentration of 0.3% by weight is thereby obtained.

[0061] The structure of the tubules is characterized by thecomplementary use of central X-ray and neutron scattering (reciprocalspace of the wavenumber transfer moment) and by transmission electronmicroscopy (real space with three spatial dimensions).

[0062] Central X-ray scattering has the advantage of irradiating a largesample volume, and of thus obtaining a response which is a statisticalaverage of the probed nanostructures on this volume. The response is acurve of the scattered intensity versus the scattering angle which hasup to seven characteristic oscillations and a characteristic decay.

[0063] These components may be accounted for with theoretical modeling,if a geometrical model of hollow tubes with an external diameter of 520angstroms and with an internal diameter of 490 angstroms is used.

[0064]FIG. 1 illustrates typical X-ray scattering from a system oftubules (scattered intensity versus scattering angle).

[0065] In this figure, several experimental oscillations (curve with dotand dash lines) are seen, which the simplified theoretical model of ahollow cylinder (curves in solid lines and dashed lines) reproduces intheir positions.

[0066] Unambiguous confirmation of the existence of these tubules isbrought by transmission electron microscope photographs. The apparatusand experimental procedure used are selected for direct observation ofthe structures without any manipulation of the system. The electroniccontrast of the system deposited as a very thin layer (about 3,000angstroms) is utilized on a cooled stage of a specialized microscope.This method is described by T. Ruiz, I. Erk and J. Lepault in Biol.Cell, 1994, 80, p 203-210 [9], for observing brittle biologicalstructures.

[0067]FIG. 2 is an electron micrograph showing the quasi-exclusivepresence of long hollow cylinders. The walls and ends of these tubulesmay perfectly be seen in this figure. The external and internaldiameters are 520 angstroms and 490 angstroms respectively.

[0068] The possibilities of orientation of the tubules are now examinedin the case of the 0.3% tubule solution and in the case of a solution oftubules at a concentration of 2.4% obtained in the same way.

[0069] For this purpose, the aqueous solution containing the tubules issubmitted to a shear or elongational stress (γ) in order to orientatethe tubules.

[0070] The value of this minimum shear stress is small, typically ofabout 100 s⁻¹ for a concentration of about 2%. Superior results areobtained from stretched fibers (elongation) of a more concentratedhighly viscous solution (7%). Characterization is performed by centralX-ray scattering.

[0071] For this purpose, a sample of tubules is placed in the 1 mm gapformed by two coaxial cylinders, with the rotation of the externalelement generating a shear in the gap. The gap is struck by an X-raybeam, whereby the scattered intensity thereof by a two-dimensionaldetector enables the degree of orientation of the tubules to beanalyzed. If the tubules are randomly distributed in the sample'svolume, the scattering signal becomes apparent on the detector 2 d byiso-intensity lines which are circular. In this rest configuration, thescattering signature is then re-discovered with the aforementionedcharacteristic oscillations.

[0072] When the sample is stretched, the positions of the tubules becomecorrelated and the scattering signal is more or less strongly deformedaccording to the degree of orientation. Thus, one passes from circulariso-intensity lines to elliptical lines then to a line joining thescattering spots, this is what is observed with the alkaline tubulesolution.

[0073] The orientability may also be apprehended or monitored by moretraditional viscoelasticity measurements.

[0074] In this case, regardless of the concentration used, the level ofmutual interactions of the tubules is measured, i.e., the stress vs.shear rate curves and the changes in viscosity when the shear rateincreases. Under these conditions, fluidification of the system isobserved due to these orientation effects. These measurements may becarried out with a simple rheometer.

[0075] The nanotubules may be identified by their dimensions (external,internal diameters and thickness of the wall) and their physico-chemicalcharacteristics, in particular the parameters describing theirviscoelasticity (flow curves, threshold stresses, elasticity andviscosity).

[0076]FIG. 3 depicts an example of a rheological imprint of the systemand illustrates the stress vs. shear rate curves, for two tubulesolutions with a concentration of 0.3% (curve 1) and a concentration of2.4% (curve 2). The threshold stresses (σ1 and σ2) of both solutions arethereby obtained. The transverse dimensions of the tubules and theirrheological features are set by the chemical formula of the constituent,lithocholic acid, and of the type of liquid used.

CITED REFERENCES

[0077] [1] Bong, D. T., Clark, T. D. Granja, J. R. and Ghadiri, M. R.(2001), Angew. Chem. Int. Ed., 40, 988-1011.

[0078] [2] Trau M., Yao N., Kim E., Xia Y., Whitesides, G. M. and AksayI. A. (1997), Nature, 390, 674-676.

[0079] [3] Van Nostrum, C. F. (1996), Adv. Mater., 8, 1027-1030.

[0080] [4] Dekker, C. (1999), Physics Today, 52, 22-28.

[0081] [5] Schnur, J. M. (1993), Science, 262, 1669-1676.

[0082] [6] Schnur, J. M. and Shashidhar, R. (1994), Adv. Mater., 6,971-974.

[0083] [7] Gu, W., Lu, L., Chapman, G. B. and Weiss, R. G. (1997) J.Chem. Soc., Chem. Commun., 543-544.

[0084] [8] Jung, H. J., Amaike, M. and Shinkai, S. (2000), J. Chem.Soc., Chem. Commun., 2343-2344.

[0085] [9] Ruiz et al. in Biol. Cell, 1994, 80, 203-210.

1. A method for preparing organic nanotubules in an aqueous medium,comprising the following steps: a) preparing a basic aqueous solutionwith a pH from 12 to 14, preferably from 12 to 13.5, b) adding to thesolution, an organic compound of formula:

wherein R¹ is a C₁₇-C₂₀ polycyclic radical with fused rings, optionallyincluding one or more C₁₇-C₂₀ alkyl substituents, R² is a C₃-C₂₀ linearor branched alkylene group, and R³ represents a hydrogen atom, a C₁-C₂₀alkyl or C₆-C₃₀ aromatic group, and c) submitting the solution tostirring for sufficient time in order to form stable tubules of theorganic compound in the solution.
 2. The method according to claim 1wherein R² is the group:


3. The method according to any of claims 1 and 2, wherein R¹ is apolycyclic group of formula:

which may include one or more C₁ to about C₂₀ alkyl substituents.
 4. Themethod according to claim 1 wherein the organic compound is lithocholicacid of formula:


5. The method according to any of claims 1 to 4, wherein the aqueoussolution is a sodium hydroxide solution.
 6. The method according toclaim 4, wherein the aqueous solution is a sodium hydroxide solutionwith a pH of about 12.5.
 7. Nanotubules of an organic compound offormula:

wherein R¹ is a C₁₇-C₂₀ polycyclic radical with fused rings, optionallyincluding one or more C₁-C₂₀ alkyl substituents, R² is a C₃-C₂₀ linearor branched alkylene, R³ represents a hydrogen, a C₁-C₂₀ alkyl or aC₆-C₃₀ aromatic group.
 8. The nanotubules according to claim 7, whereinR¹ is a polycyclic group of formula:

which may include one or more about C₁-C₂₀ alkyl substituents. 9.Nanotubules of lithocholic acid in an aqueous medium.
 10. Use of organicnanotubules obtained by the method according to any of claims 1 to 6 formanufacturing inorganic nanotubules by mineralization.
 11. Use oforganic nanotubules obtained by the method according to any of claims 1to 6 for manufacturing field emission cathodes by metallization ofnanotubules.
 12. Use of organic nanotubules obtained by the methodaccording to any of claims 1 to 6 for manufacturing biosensors.